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Introduction to Rabies Virus-based Monosynaptic Tracing

The core idea of the system is to infect the cell population of interest with a deletion-mutant tracing virus that lacks the ability to infect neighboring cells. For example, G-deleted rabies lacks the viral glycoprotein (G) gene, which is not required for the transcription or replication of the genome within infected cells, but is required for transynnaptic spread (Wickersham et al., 2007a, Wickersham et al., 2007b). Thus, G-deleted rabies can infect a single "starter" cell, but new virus cannot spread to any neighboring cells. To enable monosynaptic spread of this deletion-mutant rabies, the essential G protein is provided to the starter cell in trans, via delivery by an AAV. Since this starter cell now has all the viral genes present, the virus can spread from this cell to synaptically connected cells (meaning, it can spread to cells that are projecting to the starter cell). Once in the secondarily infected cells that lack the G protein, the virus can replicate but the newly formed viruses are unable to infect any neighboring cells. Thus, through complementation, G-deleted rabies virus can spread to monosynaptically connected cells.

To further refine the targeting of G-deleted rabies to specific starter cells, the tropism of this virus was modified so that it could only infect a genetically specified neuronal population. This is done using the envelope protein of (EnvA) of leukosis virus. EnvA can direct virus infection specifically into cells that express the TVA viral receptor. Thus, by pseudotyping the rabies virus with EnvA, only neuronal cells engineered to express TVA will be infected.

Schematic depicting steps for using AAV and modified rabies virus (RABV) for monoclonal neuronal tracing. (1) AAV encoding TVA and G-protein is delivered to the starter cell. (2) The starter cell now expresses TVA and G-protein. EnvA-pseudotyped RAVB is delivered to the starter cell, and specifically targets the starter cell via the EnvA receptor. RABV is able to replicate in the starter cell using the G-protein, which is supplied in trans. (3) Nascent G-protein coated RAVB is released from the starter cell and free to infect neighboring cells via G-protein receptors, which are naturally expressed at presynaptic nerve terminals. (4) Neurons projecting to the starter cell are infected with G-protein coated RABV. These projection neurons do not express G-protein, and thus do not give rise to G-protein coated RAVB.